·論著·

炎性因子對(duì)人胰腺癌PaTu8988細(xì)胞NF-κB及Hedgehog通路成員表達(dá)的影響

王玉瓊丁佳寅諸嫻吳紅玉金晶滿曉華高軍李兆申

【摘要】目的探討腫瘤壞死因子 α(TNF-α)和白介素1β(IL-1β)對(duì)人胰腺癌PaTu8988細(xì)胞核因子κB(NF-κB)及Hedgehog(HH)通路成員Shh、SMO、Gli1、SuFu基因表達(dá)的影響。方法分別應(yīng)用TNF-α和IL-1β刺激人胰腺癌PaTu8988細(xì)胞48 h，以未處理細(xì)胞作為對(duì)照組。采用實(shí)時(shí)定量RT-PCR和蛋白質(zhì)印跡法檢測(cè)各組細(xì)胞NF-κB及Shh、SMO、Gli1、SuFu的mRNA和蛋白表達(dá)，采用流式細(xì)胞儀檢測(cè)各組細(xì)胞的凋亡。結(jié)果TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞的NF-κB mRNA表達(dá)量分別為9.92±0.78、7.74±0.32、1.01±0.08；Gli1 mRNA為7.25±0.45、5.74±0.33、1.00±0.06；Shh mRNA為3.60±0.36、4.33±0.45、1.00±0.04；SMO mRNA為1.03±0.15、1.07±0.16、1.01±0.06；SuFu mRNA為0.88±0.14、0.96±0.13、1.01±0.05。其中TNF-α刺激組、IL-1β刺激組的NF-κB、Gli1、Shh mRNA表達(dá)量均較對(duì)照組顯著增加，差異有統(tǒng)計(jì)學(xué)意義(P值<0.05或<0.01)。兩刺激組的NF-κB、Gli1、Shh蛋白表達(dá)量也較對(duì)照組增高，差異具有統(tǒng)計(jì)學(xué)意義(P值均<0.05)。TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞凋亡率分別為(17.40±2.87)%、(11.05±1.34)%、(49.90±2.96)%，TNF-α刺激組、IL-1β刺激組的細(xì)胞凋亡率均較對(duì)照組顯著減少，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。結(jié)論TNF-α和IL-1β可激活PaTu8988細(xì)胞的NF-κB和HH信號(hào)通路的Shh、Gli1基因的表達(dá)，減少細(xì)胞的凋亡。

【關(guān)鍵詞】胰腺腫瘤;Hedgehog蛋白質(zhì)類；信號(hào)傳導(dǎo)；炎癥;NF-κB

DOI:10.3760/cma.j.issn.1674-1935.2015.01.005

基金項(xiàng)目：國(guó)家自然科學(xué)基金(81272663，30910103911)；國(guó)家科技支撐計(jì)劃(2006BAI02A12)；上海市重點(diǎn)科技攻關(guān)項(xiàng)目(11441901800)

收稿日期：(2014-08-21)

Pro-inflammatory cytokines inhibit apoptosis of pancreatic ductal adenocarcinoma cells via both NF-κB and Hedgehog signaling pathwaysWangYuqiong,Dingjiayin,ZhuXian,WuHongyu,JinJing,ManXiaohua,GaoJun,LiZhaoshen.DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai210043,China

Correspondingauthor:GaoJun,Email:gaojunaaa@gmail.com;LiZhaoshen,Email:zhsli@81890.net

Abstract【】ObjectiveTo investigate the effect of TNF-α and IL-1β on the expression of human pancreatic cancer PaTu8988 NF-κB and Hedgehog (HH) signaling pathways members Gli1, Shh, SMO, SuFu. MethodsPancreatic cancer cell line PaTu8988 was treated with TNF-α and IL-1β, respectively, for 48 h, and cells without treatment were control group. Then the protein and mRNA expression of NF-κB and Shh, SMO, Gli1, SuFu was determined by RT-PCR and Western blot; apoptosis of cells were tested by flow cytometry. ResultsThe expressions of NF-κB mRNA in PaTu8988 cells in TNF-α group, IL-1β group, control group were 9.92±0.78, 7.74±0.32, 1.01±0.08; and the expressions of Gli1 mRNA were 7.25±0.45, 5.74±0.33, 1.00±0.06; and the expressions of Shh mRNA were 3.60±0.36, 4.33±0.45, 1.00±0.04; and the expressions of SMO mRNA were 1.03±0.15, 1.07±0.16, 1.01±0.06; and the expressions of SuFu mRNA were 0.88±0.14, 0.96±0.13, 1.01±0.05. The expressions of NF-κB, Gli1,

作者單位：210043第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院消化內(nèi)科(王玉瓊、諸嫻、丁佳寅、吳紅玉、金晶、滿曉華、高軍、李兆申)；解放軍411醫(yī)院分院內(nèi)科(王玉瓊)

通信作者：高軍，Email：gaojunaaa@gmail.com;李兆申，Email：zhsli@81890.net

Shh mRNA in TNF-α group, IL-1β group were significantly higher than that in control group, and the difference between the two groups was statistically significant (P<0.05). The expressions of corresponding proteins were consistent with the expressions of mRNA. The apoptosis rates in TNF-α group, IL-1β group, control group were (17.40±2.87)%, (11.05±1.34)%, (49.90±2.96)%, and the apoptosis rates in TNF-α group, IL-1β group were significantly lower than that in control group, and the difference between the two groups was statistically significant (P<0.01). ConclusionsTNF-α and IL-1β can activate the expression of NF-κB and Shh, Gli1 in human pancreatic cancer PaTu898, and decrease apoptosis.

【Key words】Pancreatic neoplasms;Hedgehog proteins;Signal transduction;Inflammation;NF-kappa B

流行病學(xué)研究發(fā)現(xiàn)，高達(dá)15%的腫瘤的發(fā)生、發(fā)展與感染引起的炎癥相關(guān)[1-2]。轉(zhuǎn)錄因子NF-κB是炎癥因子中的重要成員，正常情況下與inhibitor kappa B(IκB)結(jié)合處于無活性狀態(tài)，炎癥時(shí)IκB激酶(IκB kinase, IKK)復(fù)合物可被腫瘤壞死因子-α(TNF-α)和白介素1β(IL-1β)等激活，通過泛素-蛋白酶途徑降解，從而釋放出NF-κB，從胞質(zhì)轉(zhuǎn)移到胞核內(nèi)，進(jìn)而調(diào)控下游基因表達(dá)[3-4]。研究證實(shí)，NF-κB參與胰腺癌的發(fā)生[5-6]。Hedgehog(HH)信號(hào)通路在胚胎發(fā)育及胰腺癌等腫瘤發(fā)生和發(fā)展中起重要作用。HH信號(hào)通路主要由配體sonic hedgehog(Shh)、跨膜蛋白受體smoothened (SMO)、核轉(zhuǎn)錄因子glioma-associated oncogene (Glis)和抑制因子suppressor of fused(SuFu)等組成。一些信號(hào)通路也可通過影響SuFu、SMO而活化HH通路[7]。

近期研究發(fā)現(xiàn)，在彌漫性大B細(xì)胞淋巴瘤中，HH信號(hào)通路和NF-κB共同異常增多[7]。為此，本研究應(yīng)用TNF-α、IL-1β刺激胰腺癌細(xì)胞株P(guān)aTu8988，檢測(cè)細(xì)胞受刺激后NF-κB及HH信號(hào)通路成員表達(dá)及細(xì)胞凋亡的變化，探討其作用機(jī)制。

材料與方法

一、細(xì)胞培養(yǎng)及分組

胰腺癌細(xì)胞株P(guān)aTu8988由上海長(zhǎng)海醫(yī)院消化內(nèi)科實(shí)驗(yàn)室保存。常規(guī)培養(yǎng)、傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞，以5×105個(gè)細(xì)胞密度接種于10 cm培養(yǎng)皿，共接種3皿，加入含10%胎牛血清(FBS)的DMEM培養(yǎng)液(含青霉素、鏈霉素各10 U/ml)置37℃、5% CO2條件下培養(yǎng)。細(xì)胞貼壁后換含1% FBS的DMEM培養(yǎng)液(含青霉素、鏈霉素各10 U/ml)“饑餓”12 h，分別以10 ng/ml的人重組TNF-α、5 ng/ml的人重組IL-1β刺激PaTu8988細(xì)胞48 h，收集細(xì)胞。以未處理的細(xì)胞作為對(duì)照組。人重組TNF-α及IL-1β均購(gòu)自B&D公司。

二、蛋白質(zhì)印跡法檢測(cè)蛋白表達(dá)

按蛋白提取試劑盒(Thermo公司)說明書分別提取細(xì)胞胞質(zhì)蛋白及胞核蛋白，以二喹啉甲酸法測(cè)蛋白濃度。取待測(cè)樣本30 μg上樣，常規(guī)行蛋白質(zhì)印跡法檢測(cè)Shh、SMO、Gli1、SuFu、NF-κB蛋白表達(dá)，分別以Histone、GAPDH為內(nèi)參。一抗的工作濃度分別為1∶3 000、1∶1 000、1∶1 000、1∶1 000、1∶200、1∶2 000、1∶2 000，二抗工作濃度1∶5 000，最后ECL發(fā)光，X片曝光、顯影、定影。通過凝膠成像系統(tǒng)掃描各條帶的灰度值。以目的條帶與內(nèi)參條帶的灰度值比表示蛋白相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次，取均值。

三、實(shí)時(shí)定量聚合酶鏈反應(yīng)(qRT-PCR)法檢測(cè)mRNA表達(dá)

收集各組PaTu8988細(xì)胞，采用Trizol(Takara公司)抽提細(xì)胞總RNA。先逆轉(zhuǎn)錄成cDNA，再行PCR反應(yīng)。Shh、SMO、Gli1、NF-κB、SuFu、Histone、GAPDH探針均購(gòu)自Life Technology公司。實(shí)時(shí)PCR反應(yīng)程序：95℃ 10 min，95℃ 10 s、60℃ 1 min，40個(gè)循環(huán)。以公式2-△△Ct計(jì)算mRNA相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次，取均值。

四、流式細(xì)胞儀檢測(cè)細(xì)胞凋亡

Annexin V/FITC 試劑盒購(gòu)自eBioscience公司，按說明書操作。收集刺激48 h的各組PaTu8988細(xì)胞，PBS沖洗3次后以200 μl Binding buffer重懸，加入3 μl Annexin V，暗室放置15 min，加6 μl碘化丙啶，混合后上流式細(xì)胞儀(美天妮公司產(chǎn)品)檢測(cè)細(xì)胞凋亡。結(jié)果用flowjo軟件分析。實(shí)驗(yàn)重復(fù)3次，取均值。

五、統(tǒng)計(jì)學(xué)處理

結(jié)果

一、TNF-α、IL-1β刺激后PaTu8988細(xì)胞Gli1、NF-κB mRNA及蛋白表達(dá)變化

TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞的NF-κB mRNA表達(dá)量分別為9.92±0.78、7.74±0.32、1.01±0.08，蛋白表達(dá)量分別為1.00±0.01、0.98±0.03、0.79±0.04，TNF-α、IL-1β刺激組的表達(dá)均較對(duì)照組顯著增加，差異有統(tǒng)計(jì)學(xué)意義(t值分別為19.19、30.11、14.55、11.44，P值均<0.05，圖1)。TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞的Gli1 mRNA表達(dá)量分別7.25±0.45、5.74±0.33、1.00±0.06，蛋白表達(dá)量分別為0.96±0.05、0.93±0.04、0.70±0.04，TNF-α、IL-1β刺激組的表達(dá)均顯著高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(t值分別為25.11、22.07、5.51、7.22，P值均<0.05，圖1)。

圖1　對(duì)照組(1)、TNF-α刺激組(2)、IL-1β刺激組(3)PaTu8988細(xì)胞胞核內(nèi)Gli1、NF-κB蛋白表達(dá)

二、TNF-α、IL-1β刺激后PaTu8988細(xì)胞Shh、SMO、SuFu mRNA及蛋白表達(dá)變化

TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞Shh mRNA的表達(dá)量分別為3.60±0.36、4.33±0.45、1.00±0.04，蛋白表達(dá)量為0.85±0.05、0.75±0.06、0.60±0.04，兩刺激組的表達(dá)均較對(duì)照組顯著增加，差異有統(tǒng)計(jì)學(xué)意義(t值分別為13.35、13.88、45.08、6.99，P<0.05或<0.01，圖2)。TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞SMO mRNA的表達(dá)量分別為1.03±0.15、1.07±0.16、1.01±0.06，蛋白表達(dá)量為0.73±0.04、0.70±0.02、0.72±0.01；SuFu mRNA的表達(dá)量分別為0.88±0.14、0.96±0.13、1.01±0.05，蛋白表達(dá)量為0.72±0.01、0.74±0.05、0.73±0.00，兩刺激組與對(duì)照組的差異均無統(tǒng)計(jì)學(xué)意義(t值分別為0.16、0.57、0.63、2.61、1.52、0.63、0.82、0.32，P值均>0.05，圖2)。

三、TNF-α、IL-1β 刺激后PaTu8988細(xì)胞凋亡變化

TNF-α刺激組、IL-1β刺激組、對(duì)照組PaTu8988細(xì)胞凋亡率分別為(17.40±2.87)%、(11.05±1.34)%、(49.90±2.96)%(圖3)。TNF-α、IL-1β刺激組的細(xì)胞凋亡均較對(duì)照組顯著減少，差異有統(tǒng)計(jì)學(xué)意義(t值分別為19.98、29.97，P值均<0.01)。

圖2　對(duì)照組(1)、TNF-α刺激組(2)、IL-1β刺激組(3)PaTu8988細(xì)胞胞質(zhì)內(nèi)Shh、SMO、SuFu蛋白的表達(dá)

圖3對(duì)照組(3A)、TNF-α刺激組(3B)、IL-1β刺激組(3C)PaTu8988細(xì)胞的凋亡圖

討論

在對(duì)彌漫性大B細(xì)胞淋巴瘤的研究中發(fā)現(xiàn)，NF-κB可激活HH通路，上調(diào)SMO的表達(dá)[7]。另有研究[8-14]報(bào)道，包括EGF/EGFR、Wnt/β-鏈蛋白、TGF-β1、TGF-β R在內(nèi)的多個(gè)信號(hào)通路可影響正常細(xì)胞和腫瘤細(xì)胞SuFu的作用而調(diào)控Glis的表達(dá)和(或)穩(wěn)定性，導(dǎo)致HH通路活化，促進(jìn)HH靶基因表達(dá)。

胰腺癌發(fā)病過程極為復(fù)雜，有多種信號(hào)系統(tǒng)參與。NF-κB和HH信號(hào)通路是其中的兩個(gè)重要系統(tǒng)，它們的相互作用越來越受到學(xué)者的關(guān)注。Nakashima等[15]研究發(fā)現(xiàn)，TNF-α、IL-1β等通過激活NF-кB誘導(dǎo)Shh過度表達(dá)，激活HH信號(hào)通路，且NF-κB p65與Shh在胰腺癌組織中的表達(dá)呈顯著正相關(guān)，阻斷NF-κB可以抑制Shh mRNA的表達(dá)。

本研究結(jié)果顯示，TNF-α、IL-1β刺激胰腺癌PaTu8988細(xì)胞后，NF-κB表達(dá)上調(diào)，同時(shí)HH通路的Shh、Gli1基因表達(dá)也上調(diào)，而SMO及SuFu表達(dá)量無變化，提示炎癥因子的刺激可同時(shí)激活NF-κB與HH通路，兩條通路存在一定的相關(guān)性，NF-κB可能通過Gli1入核增多而激活HH通路。本研究結(jié)果還顯示，炎癥因子刺激可抑制PaTu8988細(xì)胞的凋亡。

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(本文編輯：屠振興)